Gangliosides are Transported from the Plasma Membrane to

Bioscience Reports, Vol. 19, No. 4, 1999
Gangliosides are Transported from the Plasma Membrane
to Intralysosomal Membranes as Revealed by
Immuno-Electron Microscopy
W. Mobius,1 V. Herzog,2 K. Sandhoff,1 and G. Schwarzmann1,3
A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its
transport along the endocytic pathway of cultured fibroblasts by immuno-electron
microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal
membranes. Our studies show that during endocytosis the biotin-GM1 was transported
to these intraendosomal and intralysosomal membranes. These observations support the
hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal
vesicles.
KEY WORDS: Biotin-GM1; immuno-electron microscopy; ganglioside transport;
endocytosis; intralysosomal membranes.
ABBREVIATIONS: biotin-GM1, Galactopyranosyl/3-3(2-acetamido-2-deoxy)-galactopyranosylB-4-(N-biotinyl-e-amidocaproyl-)/neuraminyla-3 galactopyranosyl]B-4 glucopyranosylJ3-l-(2S,3R,4E)-2-[l-14C]-octadecanamido-4-octadecen-l,3-diol. BMP, bis(monoacylglycero)phosphate; BSA, bovine serum albumin; BSA-Au20, (20 nm) gold particles of
20 nm diameter coated with BSA; CF, cationized ferritin; cps, centipoises; CWFS-gelatin,
cold water fish skin gelatin; DME, Dulbecco’s modified Eagle medium; FCS, fetal calf
serum; EGF, epidermal growth factor; GSL, glycosphingolipids; HEPES, N-[2-hydroxyethylJpiperazine-W-p-ethanesulfonic acid]; LAMP-1, lysosome-associated membrane protein 1; LIMP, lysosome-integral membrane protein; LR-Gold, London Resin-Gold; MPR,
mannose-6-phosphate receptor; OD, optical density at 520 nm; PBS, phosphate buffered
saline; PLT, progressive lowering of temperature; SAPs sphingolipid activator proteins.
INTRODUCTION
Gangliosides are ubiquitous in vertebrate tissue and are highly abundant in neuronal
plasma membranes. The lipophilic ceramide moiety is embedded in the outer leaflet
of the lipid bilayer whereas the hydrophilic sialooligosaccharide residue faces the
extracellular space and contributes to the glycocalix (Hakomori, 1981, Ledeen, 1985,
Wiegandt, 1985).
1
Kekule-Institute fur Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany. E-mail: schwarzmann@uni-bonn.de
2
Institut fur Zellbiologie der Universitat Bonn, 53121 Bonn, Germany.
3
To whom correspondence should be addressed.
307
0144-8463/99/0800-0307$16.00/0 © 1999 Plenum Publishing Corporation
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Mobius, Herzog, Sandhoff, and Schwarzmann
Gangliosides as well as other membrane constituents are removed from the
plasma membrane by endocytosis and are either recycled to the plasma membrane
or transported to lysosomes for degradation. Important sorting events occur at the
stage of early endosomes (Mellman, 1996): Recycling membrane components are
concentrated in the tubular extensions of early endosomes and subsequently included
in recycling vesicles while soluble components like dissociated ligands remain in the
vesicular portion of the endosome and are transported to lysosomes (Geuze et al.
1987). It is argued that specialized domains enriched in membrane components,
destined for lysosomal degradation, invaginate and bud off into the endosomal
lumen thus forming intraendosomal vesicles and other membrane structures that are
delivered to lysosomes and become intralysosomal vesicles (Harding et al., 1985,
Hopkins et al., 1990, Furst and Sandhoff, 1992, Futter et al., 1996).
Gangliosides like other glycosphingolipids (GSL) are degraded in a stepwise
manner by specific acid exohydrolases. Some of these enzymes need the assistance
of small glycoprotein cofactors (sphingolipid activator proteins, SAPs) for the degradation of GSL with short oligosaccharide head groups (Sandhoff and Klein, 1994).
Degradation of GSL occurs mainly in lysosomes. Therefore, if GSL originating from
the plasma membrane are largely included in luminal vesicles of endocytic organelles
then they would reside mainly in intralysosomal membranes and less in the limiting
membrane of lysosomes following endocytic membrane flow. This may illustrate
how membrane lipid substrates could be selectively degraded while keeping intact
the limiting lysosomal membrane which is protected by lactosamine structural
elements of lysosomal integral membrane protein (LIMP) and lysosomal associated
membrane protein (LAMP). It has thus been postulated that intralysosomal membrane are the likely place of GSL degradation (Furst and Sandhoff, 1992, Sandhoff
and Kolter, 1996). This postulate is supported by the fact that lysosomal sphingolipid storage disorders are characterized by a massive accumulation of vesicles and
membranes in the lumen of late endosomes and lysosomes (Suzuki and Chen, 1968,
Bradova et al., 1993, Burkhardt et al., 1997).
To verify this hypotheis we used a biotin-labeled exogenous ganglioside (biotinGM1) to study its intracellular distribution after many hours of endocytic membrane
flow. This GM1 derivative differs solely from GM1 in that it contains a spacerlinked biotinyl residue in place of an acetyl moiety of its sialic acid portion (Fig. 1).
A major advantage of this approach is the direct visualization of a membrane tracer
that can be distinguished from the endogenous lipids. Moreover, the biotin-tag
allows a reliable localization of the exogenously applied and labeled ganglioside
which is often difficult to achieve with anti-ganglioside antibodies as shown by
Schwarz and Futerman (1997). Metabolic studies in fibroblasts and in vitro degradation of a [C-14]-labeled biotin-GMl showed that the GM1 derivative was partially
degraded to the corresponding GM2 and GM3 derivatives. Further degradation by
sialidase was inhibited by the biotin residue (Albrecht et al., 1997). Both, the resistance of the ganglioside derivative against further degradation and the occurrence of
catabolic products, indicating that the biotin-GM1 reached degradative compartments, make biotin-GM1 a valuable tool for studies of the endocytic membrane
flow. By electron microscopy we demonstrate that biotin-GM1, after prolonged
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309
Fig. 1. Structures of ganglioside GM1 and a biotin-labeled derivative of GM1 (biotin-GM1). Asterisk
denotes position of radiocarbon.
endocytosis, is predominantly localized to intraendosomal and intralysosomal membranes as judged from electron microscopy studies.
MATERIALS AND METHODS
Endocytic Tracer Studies
Fibroblast monolayers were grown in 8 cm2 Petri dishes to confluency. Lysosomes were preloaded with BSA-Au20 (20 nm gold particles, prepared according to
Frens (1973) and Handley (1989) and coated with BSA) by a pulse period of 18 h at
37°C in DME containing 0.3% fetal calf serum (FCS) and BSA-Au20 at OD 2, and
a 120 h chase period in DME containing 10% FCS. Cationized ferritin (CF) (Sigma,
Deisenhofen, Germany) was used as secondary endocytic tracer at a concentration
of 0.5 mg/ml in serum free DME. Cells were incubated with the CF-containing
medium for 10 min at 4°C and then warmed to 37°C after adding the same volume
of DME supplemented with 0.6% (FCS) to the incubation medium. Cells were
allowed to take up CF for 3 h, then washed thoroughly and fixed with 1% formaldehyde and 0.5% glutaraldehyde in 0.2 M HEPES pH 7.2. After postfixation with
1% OsO4 for 20 min at 4°C cell monolayers were embedded in Epon (Poly Bed 812,
Polysciences, Warrington, USA). Blocks were sectioned parallel to the substrate and
sections were viewed at 80 kV with a Philips CM 120 electron microscope (Philips,
Eindhoven, The Netherlands).
Immunolabeling of Biotin-GM1
Incubation media containing biotin-GM1 were prepared as described previously
(Albrecht et al., 1997). For preembedding immunolabeling human skin fibroblasts
grown as monolayers in 8cm2 Petri dishes were incubated with the biotin-GM1
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Mobius, Herzog, Sandhoff, and Schwarzmann
analogue at a concentration of 10 mM in DME supplemented with 0.3% FCS at
37°C for 72 h. For controls, fibroblasts were incubated in the above medium lacking
the biotin-GM1 analogue. After fixation with 1% formaldehyde and 0.5% glutaraldehyde in 0.2 M HEPES pH 7.2 remaining aldehydes were reduced with 1.0%
NaBH4 followed by blocking (5% BSA, 0.2% cold water fish skin (CWFS)-gelatin
in PBS) and an overnight-incubation with goat anti-biotin antibodies conjugated
to ultra-small gold (GP-US, 1:100, Aurion, Wageningen, The Netherlands). Cell
monolayers were washed thoroughly, fixed with 0.5% glutaraldehyde and embedded
in Epon after postfixation with 1% OsO4. Blocks were sectioned parallel to the
substrate, sections were silver-enhanced for 20min according to Danscher (1981)
and viewed with a Zeiss 109 electron microscope at 80 kV (Zeiss, Oberkochem,
Germany).
For embedding in LR-Gold and for the preparation of cryosections, fibroblasts
grown to confluency in 25cm2 tissue culture flasks were incubated with 10 mM of
biotin-GM1 in DME containing 0.3% FCS for 72h at 37°C. In controls biotin-GM1
was omitted. Cells were harvested by treatment with a solution of proteinase K
(Merck, Darmstadt, Germany) 0.05 mg/ml in PBS for 3 min on ice, pelleted, fixed
with 4% formaldehyde and 0.5% glutaraldehyde in 0.2 M HEPES, pH 7.2. After
postfixation with 1% OsO4 for 10 min at 4°C pellets were embedded in LR-Gold
(Polysciences, Warrington, USA) as described (Mobius et al., 1999).
For cryosections pellets were infused with 50% polyvinylpyrrolidone (PVP10,000, Sigma) and 1.15 M sucrose in 0.1 M HEPES (modified according to
Tokuyasu, 1989) at 4°C overnight on a rotator, then placed on specimen holders
and frozen in liquid nitrogen. Cryosections were collected in a 1:1 mixture of 2%
methylcellulose (Sigma, 2% = 25 cps, 25°C) and 2.3 M sucrose according to Liou et
al. (1996). Thawed sections were washed with distilled water before immunolabeling.
For immunolabeling the following antibodies were used: goat anti-biotin antibodies conjugated to 10 nm gold particles (1:100), rabbit anti-mannose-6-phosphate
receptor (MPR) (Aurion, Wageningen, The Netherlands) antibodies (1:120, a generous gift of Bernard Hoflack, Lille, France), rabbit anti-acid phosphatase antibodies (1:200, Sigma, Deisenhoven, Germany), monoclonal mouse anti-LAMP-1
antibodies (H4A3, 1:100) and monoclonal mouse anti-LIMP antibodies (H5C6,
1:40). Both were from Developmental Studies Hybridoma Bank, Baltimore, USA.
Goat anti-rabbit (1:50) and goat anti-mouse (1:50), both conjugated to 6 nm gold
particles were used as secondary antibodies. All antibodies were diluted in 0.2 M
HEPES pH 7.2 containing 1% BSA and 0.2% CWFS-gelatin. Controls included
single-labeling for each antibody and omission of the primary antibody. LR-Gold
sections were postfixed with 2% glutaraldehyde in 0.2 M HEPES, stained with 2%
uranyl acetate. Cryosections were postfixed with 2% glutaraldehyde in 0.2 M HEPES
pH 7.2 and embedded in 1.8% methylcellulose containing 0.4% uranyl acetate
according to Griffiths et al. (1984). Sections were viewed at 80 kV with a Philips
CM 120 electron microscope.
RESULTS AND DISCUSSION
To study the intracellular transport of biotin-GMl (Fig. 1), this ganglioside
derivative was presented to cultured cells as a micellar solution in the culture media.
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311
In aqueous media gangliosides form micelles above their critical micellar concentration of 10–9M or even less (Formisano et al., 1979; Mraz et al., 1980). Previous
studies have shown that ganglioside micelles bind to cell surface proteins (Callies et
al., 1977; Radsak et al., 1982; reviewed by Saqr et al., 1993). Single ganglioside
molecules can dissociate from the adsorbed micelles and incorporate into the plasma
membrane. This is a slow process. The bound micelles can be removed by proteases
like trypsin leaving behind ganglioside molecules which have become components
of cell membranes. Using spin-labeled ganglioside analogues we have demonstrated
that at least some 70% of the incorporated ganglioside molecules intermix with other
lipids of cell membranes. The remainder could represent either ganglioside molecules
clustered in microdomains or endocytosed ganglioside micelles (Schwarzmann et al.,
1983; Schwarzmann et al., 1987). To incorporate enough molecules of biotin GM1
into cells sufficient for immunolabeling it was necessary to incubate cells with this
lipid for 72 h at 37°C. Longer incubation did not result in further incorporation.
Since the half life of late endosomes and lysosomes are not known we first studied
the endocytosis of fluid phase markers by fibroblasts in long-term experiments.
Endocytic Tracer Studies
Two different endocytic tracers were used to label late endosomes and lysosomes and to study the time course of endocytosis. For this, fibroblasts were incubated with the first endocytic tracer (BSA-Au20, 20 nm) and after a chase period of
120h with the second endocytic tracer (cationized ferritin, CF). As shown in Fig. 2
after 3 h of incubation large amounts of the freshly internalized tracer (CF) colocalized with BSA-Au20 that had been endocytosed 120h earlier. This clearly demonstrates that these multilamellar lysosomes or late endosomes are long-lasting
organelles and continue to actively receive endocytosed material. A striking feature
of this compartment is the high content of internal membranes including membrane
whorls and vesicles. As these organelles contain most of the internalized BSA-Au20
and since they are only labeled by CF after at least 1 h of incubation (data not
shown) they are operationally defined as lysosomes. Internal membranes are also
frequently observed in endosomes and lysosomes of other cell types (van Deurs et
al., 1995; Holtzman, 1989; Harding et al., 1985).
In the following experiments we examined whether biotin-GM1 was sorted into
these internal membranes during endocytosis.
Intracellular Distribution of Biotin-GM1
The distribution of biotin-GM1 in intracellular membranes after endocytosis
can be directly visualized with gold-conjugated anti-biotin antibodies. Morpholigical
studies of intracellular lipid transport often depended on fluorescent lipid analogues
and, therefore, were restricted to the limited resolution of light microscopy (van
Meer, 1989; Pagano, 1990; Putz and Schwarzmann, 1995; and Sofer et al., 1996).
For electron microscopic studies of the intracellular distribution of lipids methods
are required that maintain membrane structure and composition. We used different
approaches for the determination of the intracellular distribution of biotin-GM1:
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Fig. 2. Endocytic tracer studies: Lysosomes of human skin fibroblasts were preloaded with BSAgold. After 3 h of incubation cationized ferritin (arrowheads) clearly colocalized with gold particles
(arrows) that were internalized 120h before. The late endocytic compartments of this cell type,
identified by their gold-content, seem to be long-lasting organelles that contain large amounts of
internal membranes. er: endoplasmatic reticulum, m: mitochondria, bar = 0.5 mm.
Preembedding labeling in combination with Epon embedding, embedding of OsO4fixed samples in LR-Gold and cryosections.
Because of possible lipid loss during dehydration prior to embedding biotinGM1 was localized in fixed cells using a preembedding labeling protocol as described
in Materials and Methods. Silver enhanced gold particles indicating antibiotin antibodies were detectable in the lumen of multilamellar organeles (Fig. 3A) resembling
the multilamellar lysosomes shown by the endocytic tracer studies (Fig. 2). Cells
treated according to the preembedding labeling protocol showed a poor preservation
of their ultrastructure. However, a poorly preserved ultrastructure must be accepted
if sufficient antibody penetration for detection of intracellular antigens is to be
achieved. Another disadvantage of the preembedding labeling is the low sensitivity
and resolution of the label.
For postembedding labeling, after uptake of biotin-GM1 for 72 h, cells were
aldehyde-fixed and postfixed with OsO4 followed by embedding in LR-Gold by using
a progressive lowering of temperature (PLT)-protocol. Both, OsO4-fixation and
embedding at low temperature were applied to minimize lipid redistribution. As
shown in Fig. 3, biotin-GM1 is detectable over the membranes of multilamellar late
endosomes and lysosomes, identified by the label with antibodies against the mannose-6-phosphate receptor (MPR) (Fig. 3B) and LAMP-1 (Fig. 3C). Since the antibiotin-gold-label is always found in close membrane-association, lipid redistribution
Membrane Transport in Endocytosis
313
Fig. 3. Intracellular localization of biotin-GM1: Fibroblasts were incubated for 72 h with biotin-GM1 as described. The biotin-GM1 is detectable on luminal membranes of a multilamellar organelle by preembedding
labeling (Fig. 3A). Double immunolabeling of LR-Gold sections with goat
antibiotin-gold (10 nm) and rabbit anti-MPR (6nm gold particles, indicated by arrowheads) (Fig. 3B) and mouse anti-LAMP-1 (6nm gold particles, indicated by arrowheads) (Fig. 3C) showed that biotin-GM1 is
detectable over the membranes of late endosomes and lysosomes. Double
immunolabeling of cryosections with goat anti-biotin-gold (10 nm) and
mouse anti-LIMP (6nm gold-particles, indicated by arrowheads) (Fig.
3D) or rabbit anti-acid phosphatase (6nm gold-particles, indicated by
arrowheads) (Fig. 3E) also showed that the biotin-labeled GM1 analogue
is transported to the luminal membranes of multilamellar late endosomes
or lysosomes. bar = 0.1 mm.
during sample preparation can largely be excluded. This might have been brought
about by OsO4-fixation and dehydration at low temperature. As a disadvantage,
osmification significantly reduced antigenicity resulting in only sparse labeling for
MPR and LAMP-1.
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Mobius, Herzog, Sandhoff, and Schwarzmann
Cryosectioning provides a method that allows the localization of antigens with
a minimum of denaturation steps during sample preparation. By applying a modified
section-pick-up protocol developed by Liou et al. (1996) it was possible to localize
biotin-GM1 on cryosections due to an excellent preservation of membrane structures. Double labeling with antibiotin and antibodies against LIMP (Fig. 3D) and
acid phosphatase (Fig. 3E) localized biotin-GM1 to late endosomes and lysosomes.
In principle all three techniques applied to display the intracellular distribution of
biotin-GM1 revealed the same localization. Strikingly, most biotin-GM1 molecules
were confined to internal membrane structures of lysosomes.
Biological Implication of Our Findings
We could demonstrate that this exogenous biotinylated ganglioside derivative after
incorporation into the plasma membrane was sorted to intraendosomal and intralysosomal membranes in the course of endocytosis. This localization corresponds to
the distribution of endogenous GM1 in endocytic organelles (Parton, 1994). A similar localization has been reported for the Forssman glycolipid (van Genderen et al.,
1991). Our results support the hypothesis that GSL are sorted to intraendosomal
and intralysosomal vesicles before degradation (Furst and Sandhoff, 1992; Sandhoff
and Kolter, 1996) and that this endocytic pathway leads to ganglioside degradation
(Mobius et al., 1999).
Several findings support the idea that intralysosomal vesicles are the likely place
for the degradation of membrane components: After binding of EFG, the activated
EGF-receptor is transported to intralysosomal membranes in the process of receptor
down-regulation (Haigler et al., 1979; Beguinot et al., 1984; Hopkins et al., 1990;
Felder et al., 1990; Renfrew and Hubbard 1991; Futter et al., 1996). Cultured fibroblasts of patients affected with a sphingolipid storage disorder caused by a deficiency
of the sphingolipid activator proteins (SAPs) show large multivesicular storage
organelles consisting of late endosomes and lysosomes (Burkhardt et al., 1997).
Complementation of the medium of these SAP-precursor-deficient fibroblasts with
purified SAP-precursor completely reversed the aberrant accumulation of multivesicular structures. Recent findings by Kobayashi and co-workers showed that the
internal vesicles of endosomes are highly enriched in bis(monoacylglycero)phosphate
(BMP) (Kobayashi et al., 1998). Interestingly, in-vitro studies of the degradation of
glucosylceramide by purified glucocerebrosidase in a liposomal assay system showed
that BMP stimulated the hydrolysis of glycosylceramide up to 30-fold (Wilkening et
al., 1998). The inclusion of lipid membrane substrates in internal vesicles of lysosomes would allow their selective degradation without damagin3 the limiting membrane. However, until now the mechanism by which the limiting membrane of
endosomes buds inward to form internal vesicles is still completely unknown.
ACKNOWLEDGEMENTS
This work as made possible by a grant from the Deutsche Forschungsgemeinschaft (SFB 284).
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315
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